Abstract
Light sheet microscopy techniques have expanded with designs to address many new applications. Due to rapid advancements in computing power, camera/detector technologies, and tissue clearing techniques, light sheet methods are becoming increasingly popular for biomedical imaging applications at the cellular and tissue levels. Light sheet imaging modalities couple rapid imaging rates, low-levels of phototoxicity, and excellent signal to noise ratios, contributing to their popularity for experimental biology. However, the current major limitation of light sheet microscopy arises from optical aberrations, with the main drawback being the defocusing introduced by refractive index variations that accompany clearing techniques. Here, we propose an inexpensive and easy to build light sheet based instrumentation to overcome this limitation by optomechanically decoupling the sample scanning movement from the detection step. Our solution is relatively simple to implement and also provides increased modularity by using a swappable excitation arm. This expands the range of samples we can image on a single system, from high resolution for single cells at ? m spatial resolution, to tissues with mm spatial resolution. We demonstrate our approach, using the system to image iDISCO cleared embryos and sciatic nerves, and provide the full three-dimensional reconstruction of these objects in minutes.
Highlights
In recent years, there has been a tremendous number of new developments in optical microscopy techniques targeted at imaging different biological systems at a range of spatial and temporal scales, with each technique having its own intrinsic advantages and limitations
We focus on new developments in light sheet microscopy, known as single plane or selective plane illumination microscopy.[1,2]
Better resolution in the Z direction can only be achieved by decreasing the field of view, e.g., a light sheet thickness of 4 μm typically has a field of view of 300 μm, whereas to attain a thinner 1 μm sheet, the field of view will be reduced to 25 μm
Summary
There has been a tremendous number of new developments in optical microscopy techniques targeted at imaging different biological systems at a range of spatial and temporal scales, with each technique having its own intrinsic advantages and limitations. Applications of light sheet microscopy are rapidly increasing, especially for imaging larger samples, such as zebrafish,[14,15] fly embryos,[9,16] C. elegans,[17,18] and mouse organs or embryos.[19,20,21] Light sheet microscopes were designed to illuminate specimens with a thin light sheet resulting in intrinsic optical sectioning for fluorescence microscopy.[6,7,8,9] Recent developments have significantly increased the resolution of this imaging method using multiple angle detection to achieve isotropic reconstruction of the specimen[10,11] or via the implementation of lattice light sheet,[12] the use of Bessel beams to appreciably reduce the thickness of the illuminated plane while keeping a large field of view, and a combination of Bessel beam excitation with two-photon illumination.[13]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
